The formation of functionally distinct muscles, including the myocardium, requires the orderly expression, assembly, and interaction of a large number of proteins, many of which are members of multigene families encoding closely related but distinct isoforms. This is underscored by diseases such as familial hypertrophic cardiomyopathy which can result from mutations in at least three different muscle proteins representing both thick and thin filaments: myosin heavy chain, troponin C, or tropomyosin. This proposal focuses on the physiological significance of cardiac actin which forms the backbone of the thin filaments in the cardiomyocytes. The actin gene family includes four closely related proteins (cardiac-, skeletal-, vascular- and enteric-actin) which constitute the predominant actin in the muscles for which they are named. The principal hypotheses to be tested are that (1) the predominance of cardiac actin in the normal adult mouse heart is due to a unique physiological requirement(s) for this isoform; and (2) the structural differences among the muscle actins are related to unique functional properties of the individual members of this gene family. The applicant plans to test these hypotheses by replacing cardiac actin in the mouse heart with enteric, skeletal, or vascular actin and determining the structural or functional effects, if any, in the myocardium. If there are any effects, the applicant will initiate an analysis of the contribution of selected amino acid exchanges among the muscle actins to the observed structural, molecular, and physiological changes in the myocardium. The applicant will exploit a transgenic mouse model he has generated by ablating the cardiac actin gene in embryonic stem (ES) cells. Animals lacking cardiac actin form a functioning four-chambered heart but die within a few days of birth. The applicant has now "rescued" this lethal phenotype by ectopically expressing enteric actin in the heart using the cardiac a-myosin heavy chain (aMHC) promoter and preliminary results indicate that the hearts are enlarged and hypodynamic. The applicant proposes to alter the exons of the cardiac actin gene using double replacement or "knock-in" strategies to encode and, thus, appropriately express the alternative muscle actin in place of cardiac actin. As functional differences are revealed in the whole animal, isolated heart, or purified myofibrils, the importance of selected amino acid differences will be assessed by producing chimeric actins in the heart.